Crown gall formation on dicotyledonous plants by Agrobacterium tumefaciens is the result of the transfer and covalent integration of a small segment called transfer DNA (T-DNA) of a tumor-inducing (Ti) plasmid into the chromosomal DNA of plant cells. The transferred T-DNA is expressed in plant cells and codes for several polyadenylated transcripts. Some of the transcripts are known to be responsible for opine synthesis and for tumor growth; these latter transcripts are encoded by oncogenes. None of these transcripts have been found to be essential for T-DNA transfer. The transfer mechanism is thought to involve repeated nucleotide sequences present near the T-DNA borders. As long as these borders are present, a foreign gene can be inserted into the T-DNA of a Ti plasmid and thus engineered into the genome of a tumor cell or regenerated plant.
In addition to the T-DNA sequences, it is generally believed that another set of Ti plasmid genes, located outside the T-DNA, termed the VIR (virulence) region, play a role in the mobilization of the T-DNA from the bacterium to the plant cell. Heretofore, the presence of the crown gall (tumor) on plants infected by an Agrobacterium carrying both the T-DNA and VIR regions has been the primary means of identifying transformed plant cells. This means of identification has limited commercial utility however, because whole plants can not be regenerated from crown galls containing functional oncogenes. Thus, it would be advantageous to develop a way of introducing and expressing foreign genes in plant cells without relying on tumor genes for the selection and identification of transformed plant cells.
Presently, a variety of methods to introduce DNA into plant cells are available with varying degrees of success. These methods include the use of liposomes to encapsulate one or more DNA molecules, the contacting of plant cells with DNA (which is complexed with either polycationic substances or calcium phosphate) and protoplast fusion techniques. Currently, the preferred technique involves the utilization of Ti plasmids from Agrobacterium cells to transfer a desired gene into a plant cell. Recently, researchers at Monsanto Company have demonstrated the availability of a co-integrant Ti plasmid for use in a method for transforming plant cells. (See, Fraley and Rogers, PCT Application WO84/0219). In addition, a Ti binary vector system, developed by Hoekema et al., 1983, Nature 303:179, is known in the art.
The aforementioned T-DNA region from the Ti plasmid is available for the insertion of a desired gene which is under the control of a functional plant expression mechanism. Such chimeric genes are known in the art to express both plant- and bacterial-derived polypeptides. Prior to the present invention, a chimeric protein, that is, a heterologous gene which is fused to a portion or whole of a structural plant gene, had never been expressed in a plant cell. The present vector constructions provide for the production of such a chimeric protein and thus contributes to the continuing development of plant transformation systems.
As demonstrated for bacterial and mammalian cells, one of the primary steps in the development of efficient transformation systems is the construction of dominant selectable markers. Such markers allow cells that have acquired new genes via transformation to be selected and identified easily. The present invention provides novel expression vectors which demonstrate that the aminocyclitol antibiotic hygromycin B can be the basis of such a selection scheme for transformed plant cells.
The present invention further provides a method of selecting transformed plant cells from a background of nontransformed cells. The method allows one to add non-selectable DNA to the present vectors, transform plant cells with the modified vectors and select hygromycin resistant transformants containing this otherwise non-selectable DNA. Since transformation is a very low frequency event, such a functional test is a practical necessity for determining which cell(s), of among millions of cells, has acquired the transforming DNA.
For purposes of the present invention, as disclosed and claimed herein, the following terms are defined below.
Recombinant DNA Cloning Vectorxe2x80x94any autonomously replicating agent, including but not limited to plasmids, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
Recombinant DNA Expression Vectorxe2x80x94any recombinant DNA cloning vector into which one or more transcriptional and translational activating sequence(s) have been incorporated.
Promoterxe2x80x94the site on the DNA molecule to which RNA polymerase attaches and at which transcription is initiated.
Amino terminal region-encoding sequencexe2x80x94the region of DNA at which translation of mRNA into a polypeptide is initiated and a portion of the 5xe2x80x2 end of the resulting polypeptide is produced.
Chimeric proteinxe2x80x94a recoverable heterologous polypeptide which is synthesized from a gene containing a promoter and a portion of a homologous coding region.
Border Sequencexe2x80x94DNA sequence which contains the ends of the T-DNA.
Broad-host-range repliconxe2x80x94a DNA molecule capable of being transferred and maintained in many different bacterial cells.
Conjugationxe2x80x94the process whereby DNA is transferred from bacteria of one type to another type during cell-to-cell contact.
Crown Gallxe2x80x94a plant tumor caused by Agrobacterium tumefaciens. Ti plasmidxe2x80x94a large Agrobacterium plasmid which confers the ability to induce tumors and promotes bacterial conjugation.
Micro-Ti Plasmidxe2x80x94a plasmid capable of replication in Agrobacteria and containing DNA flanked by T-DNA borders.
Non-oncogenic Strainxe2x80x94a strain of Agrobacterium tumefaciens which is unable to induce crown gall but retains the vir functions.
Transformationsxe2x80x94the introduction of DNA into a recipient host cell that changes the genotype and results in a phenotypic change in the recipient cell.
In accordance with this invention, functional and selectable micro-Ti plasmids are disclosed. The hygromycin phosphotransferase (aphIV) gene from Escherichia coli was inserted between the 5xe2x80x2 promoter and associated amino terminal region-encoding sequence of an octopine synthetase gene and the 3xe2x80x2 terminator sequence of a nopaline synthetase gene. These constructs were assembled between T-DNA border fragments in a broad-host-range vector to form micro-Ti plasmids of the present invention.
The invention further provides a method for selecting a hygromycin-resistant recombinant-DNA containing plant cell. The method comprises:
a) conjugating a recombinant DNA expression vector of the present invention into an Agrobacterium tumefaciens strain;
b) inoculating a hygromycin-sensitive plant cell with said Agrobacterium tumefaciens containing the expression vector; and
c) selecting cells transformed by said expression vector under hygromycin resistance selective conditions.